Incorporation of heterocycles into the backbone of peptoids to generate diverse peptoid-inspired one bead one compound libraries

Article abstract of DOI:10.1021/co200195t

Combinatorial libraries of peptoids (oligo-N-substituted glycines) have proven to be useful sources of protein ligands. Each unit of the peptoid oligomer is derived from 2-haloacetic acid and a primary amine. To increase the chemical diversity available in peptoid libraries, we demonstrate here that heterocyclic halomethyl carboxylic acids can be employed as backbone building blocks in the synthesis of peptoid-based oligomers. Optimized conditions are reported that allow the creation of large, high quality combinatorial libraries containing these units.

Full text of DOI:10.1021/co200195t

Research Article
pubs.acs.org/acscombsci
Incorporation of Heterocycles into the Backbone of Peptoids to
Generate Diverse Peptoid-Inspired One Bead One Compound
Libraries
Animesh Aditya and Thomas Kodadek*
Department of Chemistry and Department of Cancer Biology, The Scripps Research Institute, Scripps Florida, 130 Scripps Way,
Jupiter, Florida 33458, United States
S
* Supporting Information
ABSTRACT: Combinatorial libraries of peptoids (oligo-
N-substituted glycines) have proven to be useful sources of
protein ligands. Each unit of the peptoid oligomer is derived
from 2-haloacetic acid and a primary amine. To increase the
chemical diversity available in peptoid libraries, we demon-
strate here that heterocyclic halomethyl carboxylic acids can be
employed as backbone building blocks in the synthesis of
peptoid-based oligomers. Optimized conditions are reported
that allow the creation of large, high quality combinatorial libraries containing these units.
KEYWORDS: peptoids, one bead one compound libraries, heterocycles, solid-phase synthesis, peptidomimetics
two previous reports indicate that incorporation of a 1,5-
disubstituted-1,2,3-triazole10 and a 1,3,5-triazine unit11 in the
peptoid backbone resulted in conformationally biased oligomers.
Moreover, heterocycles can also increase the aqueous solubility of
oligomers relative to all-carbon units.8,12 The critical role of
heterocycles in drug discovery is underscored by the fact that
more than half of known small molecule drugs contain at least
one heterocycle. Here we report novel heterocyclic halomethyl
carboxylic acids that can be used in place of the halo acetate,
allowing the creation of peptoid analogues in which a heterocycle
is incorporated into the main chain. Optimized conditions for the
creation of high quality combinatorial libraries are presented.
Furthermore, we also show that molecules derived from a single
bead of a one bead one compound library of heterocycle-modified
peptoids can be characterized by mass spectrometry.
INTRODUCTION
■
There is great interest in the discovery of potent and selective
bioactive compounds for use in medicine and biology. In recent
years, high-throughput screening has been a major contributor
to the discovery of such species. These screening campaigns are
carried out in a completely unbiased fashion if no structural
information for the target protein is available to guide library
design. In such cases, the more chemically diverse the library one
screens, the better. One source of highly diverse peptidomimetic
compounds is libraries of peptoids (N-substituted oligo-glycines).1
Peptoids have certain advantages over native peptides, including
protease insensitivity and improved cell permeability.2,3
Each unit of a peptoid is put together in two steps,4 the first
being acylation of an existing amine with an activated ester of
2-bromo (or chloro5) acetic acid, followed by displacement of
the halide with a primary amine. These are almost ideal molecules
for combinatorial chemistry,6 because the source of diversity is
a primary amine, of which thousands are readily available. In
addition, the structures of peptoids can be determined by tandem
mass spectrometry, meaning that split and pool strategies7 can
be used to create peptoid libraries without the requirement for
encoding.
It would be desirable to develop peptoid analogues that
retain the many favorable characteristics of this class of
compounds but offer increased chemical diversity and greater
conformational constraints. One strategy to accomplish this
goal would be to replace the two-carbon haloacetic acid-derived
fragment of peptoids with a more elaborate building block.8,9
Aromatic heterocycles are of particular interest as main chain
building blocks. In addition to expanding the functionality in
the main chain relative to a peptoid, these units would be expected
to introduce conformational constraints in the oligomer. Indeed,
RESULTS
■
Our initial efforts were focused toward incorporation of oxazole
and thiazole units in the peptoid backbone, deriving inspiration
from variety of natural products that display these entities.
Biosynthetic assembly of these heterocycles occurs via various
post-translational modifications on serine, threonine, and
cysteine residues in their peptide precursors. Synthetic routes
have been reported for the generation of oxazole and thiazole
amino acid building blocks for combinatorial synthesis,13−15
and several strategies to assemble these scaffolds on solid phase
have also been reported.16 However, none of these strategies
Received: November 18, 2011
Revised: January 21, 2012
Published: February 9, 2012
© 2012 American Chemical Society
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Research Article
are applicable to a submonomer, peptoid-like approach to
combinatorial library synthesis.
Therefore, we created heterocycles-containing haloacids 1
acidification yielded the respective carboxylic acids. Synthesis
of these heterocycles was performed on a 45 mmol scale
with ∼85% and ∼45% overall yields for the thiazole and the
oxazole building blocks, respectively.
and 2 (Figure 1). These heterocycles were synthesized using a
With these building blocks in hand, we turned to deriving
optimized conditions for inserting them into a peptoid chain.
A five-residue peptoid (Figure 2) was assembled on polystyrene
resin functionalized with the rink amide linker. We examined
several conditions for carboxylic acid coupling and amine
displacement (summarized in Table 1). In each case, the resin
beads were subjected to a chloranil test18 to determine the
completeness of the coupling reaction. Coupling of halomethyl
oxazole and thiazole carboxylic acids at room temperature for
3 h with N,N′-diisopropylcarbodiimide (DIC) as the coupling agent,
both in the absence and presence of additives such as HOAt were
found to be inefficient. Subsequently, we tested the utility of
standard microwave-assisted peptoid synthesis conditions19 (1 M
acid +1 M DIC in dimethylformamide (DMF) with irradiation at
100 W for 15 s) for the coupling of azole heterocycles 1 and 2 to
the N-terminus of this chain. Unfortunately, these conditions also
resulted in inefficient coupling as indicated by the chloranil test. The
sluggish nature of this coupling can be attributed to the much lower
reactivity of the activated esters of these carboxylic acids relative to
those obtained from bromo- or chloroacetic acid. Optimal coupling
of these heterocyclic carboxylic acids could only be achieved by per-
forming the DIC-mediated couplings at 70 °C, employing 10 equiv
each of 1 or 2 along with 12 equiv of DIC for a period of 5 h.
Figure 1. Synthesis of azole building blocks: (1) 2-chloromethylox-
azole-4-carboxylic acid, (2) 2-chloromethylthiazole-4-carboxylic acid.
modification of a previously published procedure17 as described
in Figure 1. Briefly, serine or cysteine methyl ester hydro-
chloride was condensed with dichloroacetonitrile in the
presence of sodium methoxide. The resulting oxazoline or
thiazoline intermediate was allowed to undergo base-catalyzed
elimination to yield the corresponding chloromethyl-azole
methyl esters. Saponification at low temperature followed by
Figure 2. Incorporation of azole heterocycles to generate a backbone modified peptoid (a) BrCH2COOH (2 M in DMF), DIC (2 M in DMF), MW
15 s, 10% power (100 W) repeated twice. (b) Primary amine (2 M in DMF), MW 30 s, 10% power (100 W) repeated three times. (c) Steps (a) and
(b) repeated four times with various amines. Different conditions for acylation with the heterocycle and subsequent displacement with the amine are
described in Table 1.
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Table 1. Optimization of Acylation and Amine Displacement Conditions to Incorporate Azole Heterocycles into the Peptoid
a
Backbone
b
f
X
S
acylation conditions
amine displacement conditions
purity
comments
2 (10 equiv) + DIC (10 equiv) in DMF, RT, 3 h.
0.5 M effective concentration of reagents,
incomplete coupling
S
2 (10 equiv) + DIC (10 equiv) + HOAt (10 equiv) in DMF,
RT, 3 h.
0.5 M effective concentration of reagents,
incomplete coupling
S
2 (2 M in DMF) + DIC (2 M in DMF) 1:1, MW, 15 s, 100 W
1 M effective concentration of reagents,
incomplete coupling
c
(10%) power , repeated 3 times
S
2 (10 equiv) + DIC (12 equiv) in DMF, 70 °C, 5 h
benzylamine (1 M in DMF), 80 °C,
91%
73%
96%
91%
0.5 M effective concentration of reagents
during acylation
12 h
O
S
1 (10 equiv) + DIC (12 equiv) in DMF, 70 °C, 5 h
benzylamine (1 M in DMF), 80 °C,
0.5 M effective concentration of reagents
during acylation
12 h
d
d
2 (10 equiv) + DIC (12 equiv) in DMF, MW , 80 °C, 20 min benzylamine (1 M in DMF), MW
100 °C, 30 min
1 M effective concentration of reagents
d
d
O
1 (10 equiv) + DIC (12 equiv) in DMF, MW , 80 °C, 20 min benzylamine (1 M in DMF), MW
1 M effective concentration of reagents
100 °C, 30 min
a
b
1 and 2 refer to azole building blocks 2-chloromethyloxazole-4-carboxylic acid and 2-chloromethylthiazole-4-carboxylic acid, respectively. Area
c
under major peak obtained in the HPLC analysis of the crude test peptoids shown in Figure 2. Microwave reaction performed in a 1000 W house
d
microwave apparatus. Microwave reaction performed in a microwave reactor (Biotage Inc.) set to fixed temperature and variable power settings.
f
Completeness of coupling was determined by a chloranil test after acylation where beads turned blue if acylation was incomplete.
Figure 3. HPLC and MALDI-MS profiles of test peptoids containing azole building blocks synthesized by microwave-assisted coupling of
heterocyclic building blocks. (A) A test peptoid containing an oxazole building block. (B) Test peptoid containing a thiazole building block.
Magnified versions of these spectra are provided in the Supporting Information.
Performing the same reaction within a temperature-controlled
microwave reactor (Biotage Inc.) at 80 °C for 20 min with 2 min of
preactivation was found to decrease the reaction time considerably.
The amine coupling reaction also proved to be more difficult
than is the case with standard peptoid synthesis. This is not sur-
prising given the reduced electrophilicity of the chloro-substituted
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different from the fragmentation trends observed with regular
peptoids and peptides. We observed that fragmentation of the
amide bond at the C-terminal side of the heterocycle was
suppressed, resulting in a complete absence or a low intensity
of the corresponding y and b fragments resulting from that
bond breakage. This unusual fragmentation trend can
potentially complicate the sequencing of these oligomers.
This means that the heterocycle-containing monomer and the
one on the C-terminal side of it must be read as a single unit in
the MS/MS spectrum. Thus, one must employ a different set of
amines with distinct molecular weights to functionalize the
azole submonomer to assign unequivocally the side chains on
the heterocycles unit and the one before it from the
fragmentation spectrum. Bearing this in mind, we synthesized
a library of heptamers containing a constant four-residue
peptoid linker appended to a peptide dimer constructed by
cysteine and 6-aminohexanoic acid at the C-terminal. The
variable three residues were diversified by using four different
amines where the sixth position was further diversified by
substituting oxazole or thiazole submonomer in place of the
usual bromoacetic acid, thus resulting in a library of 128
compounds (Figure 4). The oxazole and thiazole-containing
units were inserted using the optimized conditions mentioned
above while all of the peptoid residues were inserted using
standard microwave-assisted conditions. Twenty beads were
picked at random in a 96-well plate for quality analysis of this
library, and each bead was subjected to cleavage with
trifluoroacetic acid. The postcleavage MS analysis displayed
clean mass spectra for all compounds. MS-MS fragmentation of
the major peak obtained in each case enabled the sequencing of
all oligomers obtained from the aforementioned 20 beads,
where seven sequences appeared more than once. The design
template, amine building blocks utilized for each variable
position, and the mass spectral profile of representative
members of this library are shown in Figures 4 and 5.
Figure 4. Design of peptoid library with three diversity positions
incorporating the azole building block in the backbone. This library
was assembled on polystyrene-Rink amide resin (500 μm beads) by
utilizing microwave assisted peptoid synthesis conditions.
carbons in the heterocyles relative to a carbon adjacent to
carbonyl center. Nonetheless, we found that essentially
quantitative displacement of the chloride could be achieved
by treating the beads with primary amine (1 M in DMF) at
80 °C for 12 h or at 100 °C for 30 min in the microwave reactor.
In general, microwave-assisted couplings and amine displacement
reactions generated the resulting oligomers with a greater purity
in comparison with thermal conditions. After an additional
standard peptoid residue was added to the chain as shown in
Figure 2, the molecules were cleaved from the resin and analyzed
by HPLC and MALDI mass spectrometry (Figure 3).
We then wanted to evaluate our ability to insert these
heterocyclic building blocks into oligomeric chains in the
context of one bead one compound (OBOC) libraries created
by split and pool synthesis where the library members can still
be sequenced by tandem MS unambiguously. The MS
sequencing profiles of representative azole-modified peptoids,
shown in Figure 3, exhibit a fragmentation pattern somewhat
Figure 5. MALDI-MS and MS-MS profiles of representative members of library shown in Figure 4. These spectra were obtained from compounds
cleaved from individual beads handpicked for quality control of this library. (The fragmentation scheme along with magnified traces of these spectra
are reproduced in the Supporting Information along with spectral data for other members characterized for quality control of this library.).
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in a 1,3 configuration in the peptoid backbone, we wanted to
develop heterocyclic building blocks that can be used to exhibit
1,4 and 1,2 connectivities. To this end, we synthesized the
pyrazine 3 and furan 4 building blocks as shown in Figure 6.
These building blocks were synthesized from inexpensive
commercially available methyl substituted carboxylic acids via
N-bromosuccinimide (NBS)-mediated free radical bromination.
Briefly, 5-methylpyrazine-2-carboxylic acid was converted to its
corresponding methyl ester that was subsequently treated
with NBS in presence of AIBN as the radical initiator. The cor-
responding bromomethyl carboxylate ester was saponified at
low temperature to generate the required pyrazine building
block 3 in an overall yield of ∼43% from the starting carboxylic
acid. We envisioned utilizing the same route for generating
the furan building block 4. However, methyl 2-bromomethyl-
3-furoate20 could not be saponified at ambient temperature,
and we feared that this intermediate would be hydrolyzed to
the corresponding alcohol at higher temperatures. Instead, this
building block was obtained directly by radical bromination of
2-methyl-3-furoic acid with a yield of ∼53%. The efficacy of
these new building blocks as submonomers for incorporation
into the peptoid backbone was investigated by utilizing the
microwave-assisted conditions established for azoles earlier.
The HPLC profiles of the resulting oligomers indicated that the
microwave-assisted acylation and amine displacement con-
ditions developed earlier for the azole building blocks can be
utilized to incorporate the pyrazine and furan building blocks
efficiently as well.
Figure 6. Synthesis of pyrazine and furan building blocks for
incorporation into peptoid backbone.
Figure 7. Design of a complex peptoid-based library where
heterocyclic building blocks with various configurations are incorpo-
rated in the backbone.
The synthesis of four heterocyclic halomethyl carboxylic
acids and establishment of conditions that can be employed to
append these building blocks onto a growing peptoid chain
expands our ability to synthesize fairly diverse peptoid-derived
libraries. Design of a complex library that showcases the in-
corporation of more than one thiazole, pyrazine, and furan
building blocks in the backbone is shown in Figure 7. Bromoacetic
This encouraging outcome of incorporation of oxazole and
thiazole building blocks into the peptoid backbone prompted
us to develop other heterocyclic scaffolds. With 2,4-disubstituted
azoles in hand that orient the carboxamide and amine substitution
Figure 8. MALDI-MS and tandem MS profiles of selected members of the library shown in Figure 7. These spectra were obtained from compounds
cleaved from individual beads handpicked for quality control of this library. (The y and b fragments in the tandem MS spectra are indicated
according to the fragmentation shown in the Supporting Information.).
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acid was also included as a building block at positions where
heterocycles were incorporated to increase the overall theo-
retical diversity to 16,384 compounds in this library. This
library was assembled on polystyrene Rink amide resin (500 μm,
Rapp Polymere), and the overall quality of this library was
assessed by cleaving the oligomers from 24 beads in a 96-well
format as described earlier. Out of these, compounds obtained
from 21 beads were sequenced unambiguously, indicating that
roughly 87% of this library will yield the desired sequences. The
MS spectra for representative members are shown in Figure 8.
Diverse oligomeric libraries that display a variety of backbone
scaffolds and side-chain appendages are a promising source of
bioactive ligands. Generation of such libraries in high quality is
often challenging, primarily because the chemical reactions
utilized to generate diverse scaffolds are unable to generate the
resulting oligomers in high purity.21 In this paper, we have
reported the development of heterocyclic halomethyl carboxylic
acids as building blocks for generation of diverse backbone
scaffolds in peptoid-derived oligomers. We have also demon-
strated that these halomethyl heterocyclic carboxylic acids can
be conveniently utilized to construct peptoid-based libraries via
a submonomer approach through microwave-assisted amide
bond formation and subsequent nucleophilic substitution with
primary amines. Furthermore, this strategy has been utilized to
generate peptoid-derived libraries in the OBOC format where
the library members were sequenced by tandem mass spectro-
metry. To expand the scope of this approach, development of
other heterocyclic building blocks is currently in progress that
can further enhance the backbone diversity of oligomeric
libraries. These OBOC libraries will be screened with different
biological targets to mine for structurally diverse ligands.
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MATERIAL AND METHODS
■
A full description of the materials and methods can be found in
the Supporting Information.
ASSOCIATED CONTENT
■
(16) Biron, E.; Chatterjee, J.; et al. Solid-phase synthesis of 1,3-
azole-based peptides and peptidomimetics. Org. Lett. 2006, 8, 2417−
2420.
S
* Supporting Information
Further details on the synthesis of halomethyl heterocyclic
carboxylic acids, synthesis and HPLC profiles of test peptoids,
synthesis and MS data of OBOC library with one heterocycle
and at most two heterocycles in the backbone, and on probing
for N-acylation/alkylation of nitrogen-containing heterocycles.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(17) Hermitage, S. A.; Cardwell, K. S.; et al. An efficient, practical
approach to the synthesis of 2,4-disubstituted thiazoles and oxazoles:
application to the synthesis of GW475151. Org. Process Res. Dev. 2001,
5, 37−44.
(18) Vojkovsky, T. Detection of secondary amines on solid phase.
Pept. Res. 1995, 8, 236−237.
(19) Olivos, H. J.; Alluri, P. G.; et al. Microwave-assisted solid-phase
synthesis of peptoids. Org. Lett. 2002, 4, 4057−4059.
(20) Salimbeni, A.; Canevotti, R.; et al. N-3-substituted pyrimi-
dinones as potent, orally active, AT1 selective angiotensin II receptor
antagonists. J. Med. Chem. 1995, 38, 4806−4820.
(21) Kodadek, T. The rise, fall and reinvention of combinatorial
chemistry. Chem. Commun. (Cambridge, U.K.) 2011, 47, 9757−9763.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: Kodadek@scripps.edu.
Funding
This work was supported by a grant from the National
Institutes of Health (1RO1 GM 090294) and a contract from
the National Heart Lung and Blood Institute (NO1-HV-
00242).
Notes
The authors declare no competing financial interest.
REFERENCES
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